DESCRIPTION: The objective of this revised renewal application is the characterization of two key elements of cellular signalling and regulation pathways, protein kinases and G proteins. In the first series of studies, recent novel observations on the mechanism of activation of red cell protein kinase C will be extended and the role of this and other kinases in regulating red cell biophysical properties will be evaluated. In a second series of studies, the properties and associations of G proteins in erythroid cells will be explored. The proposed studies are designed to explore the hypothesis that the two classes of regulatory proteins, protein kinase C and G proteins, which are present in erythroid cells, play important roles in erythroid development and in mature red cell function. To achieve these objectives, the following three Specific Aims are outlined: 1. Determine the properties and functions of protein kinase C in erythroid cells. Specifically, the applicant proposes to determine which PKC isoenzymes are present in developing and mature red cells, and which of these are translocated to the membrane upon TPA stimulation.Of the isoenzymes present,he will evaluate which one(s) are responsible for membrane skeletal protein phosphorylation; he will study why calpain cleavage is required for phosphorylation of membrane substrates; what is the life time and fate of constitutively active PKM which is generated by calpain cleavage of PKC; and finally, do young, senescent or abnormal red cells have altered PKC, PKA or casein kinase content; 2. Determine the functional correlates of protein kinase activation or inhibition with biophysical properties of red cells. The effects of PKC down regulation as well as the effects of inhibitors or activators of PKC, PKA and casein kinase on red cell membrane mechanical properties and on mobility of integral proteins will be studied; and 3. The molecular basis for the association of G proteins with the red cell membrane and membrane skeletal proteins will be characterized. Quantitative assays will be developed to measure binding of purified G proteins to red cell membranes. In further studies, specific classes of erythroid membrane components involved in G protein associations will be identified. It is anticipated that these studies will lay the foundation for future investigations of G protein-receptor coupling in erythroid progenitor cells. Successful accomplishment of these goals will make a significant contribution to our understanding of the roles of these two important classes of regulatory proteins in red cell function and may also provide important and unexpected insights into their function in other types of cells as well.